Lentiviral transduction of face and limb flaps: Implications for immunomodulation of vascularized composite allografts

Angelo A.Leto Barone, Zhao Y. Zhou, Michael Warren Hughes, Ryan Park, Ruth M. Schulman, Steven Lee, Evan N. Vidar, Travis L. Shiba, Erin L. Weber, Curtis L. Cetrulo

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Background: Ex vivo introduction of an immunomodulatory transgene into a face or hand allograft may improve the risk-to-benefit ratio of vascularized composite allografts. Abrogation of the immunogenicity of the skin component of a face or hand allograft may decrease alloreactivity and permit the induction of immunologic tolerance. Proof-of-principle demonstrations of transduction of composite tissue have been established using adenoviral vectors, producing transient gene expression. The authors hypothesized that transduction, integration, and long-term expression of transgenes in a vascularized composite allograft could be achieved using lentiviral vectors. Methods: Ex vivo transduction of heterogeneous primary rat cell lines representative of a composite tissue flap's cellular architecture was performed using a luc-enhanced green fluorescent protein (eGFP) human immunodeficiency virus-1-based lentiviral vector. Ex vivo injections of rat superficial inferior epigastric artery flaps with the viral vector were performed intraarterially, intramuscularly, and intradermally. Results: Quantifiable reporter expression by flow cytometry (fluorescence-activated cell sorting) analysis and in vitro bioluminescence was observed. The luc-eGFP vector exhibited broad tropism and allowed transgene expression in relevant cell lines and throughout the flaps. Ex vivo intradermal transfection resulted in genomic integration and long-term constitutive gene expression (>150 days). Similarly, efficient intradermal transfection of face and hand flaps in a rat model corroborated this approach. Ex vivo intravascular perfusion of the vector proved inferior to intradermal injection. Conclusions: Intradermal delivery of the transgenes proved superior to intravascular perfusion. Optimization of this gene-delivery approach may allow long-term, constitutive expression of immunomodulatory proteins in face and hand allografts. Future goals include replacement of the luciferase and eGFP reporter genes with key immunomodulatory proteins.

Original languageEnglish
Pages (from-to)391-400
Number of pages10
JournalPlastic and Reconstructive Surgery
Volume129
Issue number2
DOIs
Publication statusPublished - 2012 Feb 1

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Composite Tissue Allografts
Immunomodulation
Transgenes
Extremities
Hand
Allografts
Transfection
Flow Cytometry
Perfusion
Epigastric Arteries
Gene Expression
Cell Line
Intradermal Injections
Tropism
Luciferases
Reporter Genes
HIV-1
Proteins
Skin
Injections

All Science Journal Classification (ASJC) codes

  • Surgery

Cite this

Barone, Angelo A.Leto ; Zhou, Zhao Y. ; Hughes, Michael Warren ; Park, Ryan ; Schulman, Ruth M. ; Lee, Steven ; Vidar, Evan N. ; Shiba, Travis L. ; Weber, Erin L. ; Cetrulo, Curtis L. / Lentiviral transduction of face and limb flaps : Implications for immunomodulation of vascularized composite allografts. In: Plastic and Reconstructive Surgery. 2012 ; Vol. 129, No. 2. pp. 391-400.
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abstract = "Background: Ex vivo introduction of an immunomodulatory transgene into a face or hand allograft may improve the risk-to-benefit ratio of vascularized composite allografts. Abrogation of the immunogenicity of the skin component of a face or hand allograft may decrease alloreactivity and permit the induction of immunologic tolerance. Proof-of-principle demonstrations of transduction of composite tissue have been established using adenoviral vectors, producing transient gene expression. The authors hypothesized that transduction, integration, and long-term expression of transgenes in a vascularized composite allograft could be achieved using lentiviral vectors. Methods: Ex vivo transduction of heterogeneous primary rat cell lines representative of a composite tissue flap's cellular architecture was performed using a luc-enhanced green fluorescent protein (eGFP) human immunodeficiency virus-1-based lentiviral vector. Ex vivo injections of rat superficial inferior epigastric artery flaps with the viral vector were performed intraarterially, intramuscularly, and intradermally. Results: Quantifiable reporter expression by flow cytometry (fluorescence-activated cell sorting) analysis and in vitro bioluminescence was observed. The luc-eGFP vector exhibited broad tropism and allowed transgene expression in relevant cell lines and throughout the flaps. Ex vivo intradermal transfection resulted in genomic integration and long-term constitutive gene expression (>150 days). Similarly, efficient intradermal transfection of face and hand flaps in a rat model corroborated this approach. Ex vivo intravascular perfusion of the vector proved inferior to intradermal injection. Conclusions: Intradermal delivery of the transgenes proved superior to intravascular perfusion. Optimization of this gene-delivery approach may allow long-term, constitutive expression of immunomodulatory proteins in face and hand allografts. Future goals include replacement of the luciferase and eGFP reporter genes with key immunomodulatory proteins.",
author = "Barone, {Angelo A.Leto} and Zhou, {Zhao Y.} and Hughes, {Michael Warren} and Ryan Park and Schulman, {Ruth M.} and Steven Lee and Vidar, {Evan N.} and Shiba, {Travis L.} and Weber, {Erin L.} and Cetrulo, {Curtis L.}",
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Barone, AAL, Zhou, ZY, Hughes, MW, Park, R, Schulman, RM, Lee, S, Vidar, EN, Shiba, TL, Weber, EL & Cetrulo, CL 2012, 'Lentiviral transduction of face and limb flaps: Implications for immunomodulation of vascularized composite allografts', Plastic and Reconstructive Surgery, vol. 129, no. 2, pp. 391-400. https://doi.org/10.1097/PRS.0b013e31823aeaeb

Lentiviral transduction of face and limb flaps : Implications for immunomodulation of vascularized composite allografts. / Barone, Angelo A.Leto; Zhou, Zhao Y.; Hughes, Michael Warren; Park, Ryan; Schulman, Ruth M.; Lee, Steven; Vidar, Evan N.; Shiba, Travis L.; Weber, Erin L.; Cetrulo, Curtis L.

In: Plastic and Reconstructive Surgery, Vol. 129, No. 2, 01.02.2012, p. 391-400.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Lentiviral transduction of face and limb flaps

T2 - Implications for immunomodulation of vascularized composite allografts

AU - Barone, Angelo A.Leto

AU - Zhou, Zhao Y.

AU - Hughes, Michael Warren

AU - Park, Ryan

AU - Schulman, Ruth M.

AU - Lee, Steven

AU - Vidar, Evan N.

AU - Shiba, Travis L.

AU - Weber, Erin L.

AU - Cetrulo, Curtis L.

PY - 2012/2/1

Y1 - 2012/2/1

N2 - Background: Ex vivo introduction of an immunomodulatory transgene into a face or hand allograft may improve the risk-to-benefit ratio of vascularized composite allografts. Abrogation of the immunogenicity of the skin component of a face or hand allograft may decrease alloreactivity and permit the induction of immunologic tolerance. Proof-of-principle demonstrations of transduction of composite tissue have been established using adenoviral vectors, producing transient gene expression. The authors hypothesized that transduction, integration, and long-term expression of transgenes in a vascularized composite allograft could be achieved using lentiviral vectors. Methods: Ex vivo transduction of heterogeneous primary rat cell lines representative of a composite tissue flap's cellular architecture was performed using a luc-enhanced green fluorescent protein (eGFP) human immunodeficiency virus-1-based lentiviral vector. Ex vivo injections of rat superficial inferior epigastric artery flaps with the viral vector were performed intraarterially, intramuscularly, and intradermally. Results: Quantifiable reporter expression by flow cytometry (fluorescence-activated cell sorting) analysis and in vitro bioluminescence was observed. The luc-eGFP vector exhibited broad tropism and allowed transgene expression in relevant cell lines and throughout the flaps. Ex vivo intradermal transfection resulted in genomic integration and long-term constitutive gene expression (>150 days). Similarly, efficient intradermal transfection of face and hand flaps in a rat model corroborated this approach. Ex vivo intravascular perfusion of the vector proved inferior to intradermal injection. Conclusions: Intradermal delivery of the transgenes proved superior to intravascular perfusion. Optimization of this gene-delivery approach may allow long-term, constitutive expression of immunomodulatory proteins in face and hand allografts. Future goals include replacement of the luciferase and eGFP reporter genes with key immunomodulatory proteins.

AB - Background: Ex vivo introduction of an immunomodulatory transgene into a face or hand allograft may improve the risk-to-benefit ratio of vascularized composite allografts. Abrogation of the immunogenicity of the skin component of a face or hand allograft may decrease alloreactivity and permit the induction of immunologic tolerance. Proof-of-principle demonstrations of transduction of composite tissue have been established using adenoviral vectors, producing transient gene expression. The authors hypothesized that transduction, integration, and long-term expression of transgenes in a vascularized composite allograft could be achieved using lentiviral vectors. Methods: Ex vivo transduction of heterogeneous primary rat cell lines representative of a composite tissue flap's cellular architecture was performed using a luc-enhanced green fluorescent protein (eGFP) human immunodeficiency virus-1-based lentiviral vector. Ex vivo injections of rat superficial inferior epigastric artery flaps with the viral vector were performed intraarterially, intramuscularly, and intradermally. Results: Quantifiable reporter expression by flow cytometry (fluorescence-activated cell sorting) analysis and in vitro bioluminescence was observed. The luc-eGFP vector exhibited broad tropism and allowed transgene expression in relevant cell lines and throughout the flaps. Ex vivo intradermal transfection resulted in genomic integration and long-term constitutive gene expression (>150 days). Similarly, efficient intradermal transfection of face and hand flaps in a rat model corroborated this approach. Ex vivo intravascular perfusion of the vector proved inferior to intradermal injection. Conclusions: Intradermal delivery of the transgenes proved superior to intravascular perfusion. Optimization of this gene-delivery approach may allow long-term, constitutive expression of immunomodulatory proteins in face and hand allografts. Future goals include replacement of the luciferase and eGFP reporter genes with key immunomodulatory proteins.

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